Abstract

This investigation is based on the identification of cDNA sequence information for a putative chicken homologue of the mammalian cytokine “B cell activating factor belonging to the TNF family” (BAFF) in a chicken EST-database. The cDNAs encoded for the complete open reading frame of chicken BAFF (chBAFF) including a transmembrane domain and a potential furine cleavage side. ChBAFF shows an uncommon high amino-acid sequence identity with 76% identity to human BAFF in comparison with other chicken cytokines. To investigate its biochemical and functional properties, recombinant prokaryotic chBAFF was generated in E. coli and eukaryotic chBAFF was obtained from transfected 293T cells. A polyclonal rabbit antiserum raised against His-chBAFF reacted with both, the prokaryotic and the eukaryotic cytokine and was used to quantify 293T cell derived chBAFF in an ELISA system.
Studies by northern blot analysis revealed a strong expression signal in the Bursa of Fabricius and a weak signal in the spleen, while all other tissues including thymus and gut tissue were negative. In situ hybridisation detected a wide distribution pattern for chBAFF-mRNA in all areas of the bursa of Fabricius. However, the strongest expression was seen in the medulla from bursal follikels. These studies indicate that both the bursal stroma and bursal B-cells must be considered as potential sources for chBAFF. To identify target cells for this cytokine, chBAFF receptor(s) expression was investigated in binding studies with Flag tagged chBAFF (Flag-chBAFF) and binding was analysed by flow cytometry. Receptor expression was clearly restricted to B-cells including mature B-cells from peripheral lymphoid organs as well as immature bursal B-cells. Subsequent studies on the ontogeny of chBAFF receptor expression showed a positive correlation between the cytokine receptor expression and the expression of the B-cell antigen receptor in the developing embryonic bursa.
Receptor-ligand-interaction ELISA and co-immunoprecipitation experiments demonstrated that chBAFF binds to all three mammalian BAFF receptors identified thus far. Importantly, studies of chBAFF binding to the chicken B-cell line DT40 showed that an engineered soluble form of the human BAFF-receptor BCMA (huBCMA-Fc) inhibited chBAFF binding.
The addition of 293T cell derived chBAFF to splenic lymphocyte cultures led to a significant increase in B-cell viability. This dose-dependent effect was also observed in lymphocyte cultures from ceacal tonsils and in cultures of purified (>95%) splenic B-cell preparations. While the rapid apoptosis in cultures of bursal lymphocytes could not be completely prevented, chBAFF clearly increased the survival of these immature B-cells. CFSE labelling experiments further showed that chBAFF did not induce B-cell proliferation. Therefore, it is highly probable that chBAFF, as its mammalian counterpart, is a potent inhibitor of B-cell apoptosis.
These in vitro studies were complemented by in vivo experiments with purified prokaryotic chBAFF. Daily injection of recombinant cytokine for 7 days induced a significant increase in spleen weight and B-cell frequency in spleen and caecal tonsils. Besides, the functional overexpression of chBAFF induced significantly (4-fold) increased serum IgM levels. Therefore, chBAFF does not only increase B cell numbers, it also influences their differentiation to antibody secreting cells. However, no effect was observed on the bursa of Fabricius prompting the reverse experimental approach. With the availability of a soluble cytokine specific receptor (huBCMA-Fc) in vivo knockdown studies could be performed. Daily i.p. application of huBCMA-Fc to newly hatched birds for 5 days led to decreased spleen weights and drastically reduced the B-cell frequency in spleens and caecal tonsils. In addition, bursal weights in treated birds were lower than in untreated controls. This experiment in combination with the observation of high expression levels of chBAFF-mRNA in the bursa and functional BAFF-receptor(s) on bursal B-cells strongly indicated a thus far unknown role for BAFF in early B-cell development.